Transcript Discovery Research and Cell Culture

Discovery Research and Cell Culture
The Expression Vector:
The Basis of Biotechnology Manufacturing
Escherichia coli – GFP
Escherichia coli
Green Fluorescent Protein
GFP Expression Vector
Central Dogma of Biology
Transformation and Cloning
Escherichia coli
Two Stages to Production – Both Require ATP
Cell Growth and Reproduction
Synthesis Protein of Interest
Four Levels of Organization of Protein
Structure
Denaturation
Denaturation
Proteins are the Machines and form the
Structure of Life
Proteins are used by the body for a whole host
of things, e.g. within blood (for carrying
molecules and for clotting), for digestion
(enzymes are proteins), for movement (actin
and myosin in muscle), etc. One other major
role of proteins is that of "structural proteins",
i.e. those proteins that contribute to and
sustain the integrity of the human structure.
Collagen is a structural protein.
Proteins
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Hormones like human growth hormone and insulin
Enzymes like lipase and protease
Receptors for neurotransmitters, hormones, and transferrin
Signal transduction proteins (produce cascades)
Carrier proteins (for HDL, LDL, and iron)
Membrane proteins (ion chanels)
Immunoglobulins (antibodies)
Blood Proteins: albumin, transferrin, factor VIII
DNA Transcription Factors
Actin and myosin
Hemoglobin
Structural proteins like collagen, elastin, spectrin
Animation of Signal Transduction
Pathway involving Multiple Proteins
http://www.learner.org/courses/biology/archive
/animations/hires/a_cancer1_h.html
Best Site for PROTEIN Research
www.drugbank.ca
Typical Production Process Flow
Ampule Thaw
Inoculum Expansion
(Spinner Bottles)
(Feed1)
(Feed 2)
Centrifuge
(Feed 3)
(Feed 4)
Concentration /
Diafiltration
Cryo-preservation
Chrom 3
Chrom 2
Viral Removal
Filtration
Chrom 1
Upstream/Downstream Manufacturing Overview
Large Scale Bioreactor
1 day
Media Prep
Seed Bioreactors
Working Cell
Bank
150L
Bioreactor
SubCulture
SubCulture
SubCulture
26,000L
Bioreactor
Centrifuge
Depth
Filtration
Wave
SubCulture Bag
SubCulture
Collection
Inoculum
Fermentation
24 days
31 days
Filter
Harvest
Collection
Tank
750L
Bioreactor
5,000L
Bioreactor
Column
Chromatography
Skid
Eluate
Hold
Tank
Harvest/Recovery
Viral
Inactivation
Eluate
Eluate Hold
Hold Tank
Tank
20,000L
20,000L
Column
Chromatography
Skid
8,000L
1,500L
Anion Exchange
Chromatography (QXL)
Hydrophobic Interaction
Chromatography (HIC)
Purification
Filter
Column
Chromatography
Skid
Filter
Eluate
Hold
Tank
Post-viral
Hold
Vessel
3,000L
6,000L
Protein A
Chromatography
Viral Filtering
Column
Chromatography
Skid
5,000L
Anion Exchange
Chromatography
8 days
Eluate
Hold
Tank
(QFF - Fast Flow)
Ultra Filtration
Diafiltration
Bulk
Fill
Biopharmaceutical Proteins are Parenteral
Use Aseptic Technique in Clean Rooms
Shake Flask Inoculation using BSC Class 100 (5)
Media Preparation
for Cell Growth and Protein Expression
Feeding
Doubling of Cells
and Synthesis of Protein
Media and Feeds Support each Stage
• E. coli media requires some chemicals and non-defined
components (hydrolyzed protein and yeast extract) to
grow a batch and an inducer to produce the protein of
interest. This is the cheapest medium.
• CHO cells require complex medium containing all 20
amino acids, fatty acids, and carbohydrates. Growth
media requires 10% fetal bovine serum (FBS) but can
be weaned to a serum-free medium. Most expensive
medium.
• Pichia pastoris requires chemicals and non-defined
components (hydrolyzed protein, yeast extract and
yeast nitrogen base) to grow a fed-batch and an
inducer (methanol) to express large quantities of the
protein of interest.
Escherichia coli (Prokaryot)Media
LB Broth with Arabinose
NaCl
Yeast Extract
Arabinose – The Inducer
Tryptone or Peptone
Yeast Extract
The main components of yeast extract are:
– total nitrogen content : 8 to 12 %, corresponding to a
protein content of 50 to 75 %
– amino nitrogen content : 3.0 to 5.2 %
– total carbohydrate content : 4 to 13 %
– lipid content : none or very little.
Click here to see how yeast extract is made:
http://www.eurasyp.org/public.levure.extrait.screen
Sterilizing Media/Solutions
Goal: To remove microbial contamination (bioburden)
Autoclave
Sterile Filtration (.22u pores
remove bacteria)
Sterilization by Filtration at .22u
Normal Flow Filtration : (NFF)
• Build up of retained
components on filter
surface and within filter
matrix.
• NFF is robust and easy-touse
Normal Flow (NFF)
Spinner Flasks
Placed in a CO2 incubator to provide a
controlled environment for CHO cell
scale-up
• Temperature: 37oC
• CO2: 5%
• pH: 7.2
• Agitation via Magnetic Stir Plate: 75 rpm
Shake Flasks in Shaking Incubator
A disposable WAVE bioreactor
Upstream Processing Equipment
Lab-Scale Bioreactor
3 liters
Large-Scale Bioreactor
25,000 liters
Types of Bioreactors
The Top of a 20,000 liter Commercial-Scale Bioreactor
(Process-Controlled)
Topside Head plate
1. Sample bottle assembly
2. Head plate assembly
3. Stirrer motor mount
4. Condenser air outlet
5. Condenser water outlet (from)
6. Inoculation port
7. Condenser water inlet (to)
8. CO2 overlay port
9. pH probe
10. Thermowell port
11. Mill fastener
12. Sparger
13. Feed bottle
14. Blind stopper
15. DO probe
16. 3 Feed ports
17. Harvest tube
Monitoring Growth
• The importance – The growth rate (u) and
doubling time (Td) help to determine when to
feed, when to harvest and such.
• The assays for cell growth and reproduction –
live cell counts, optical density (OD) readings,
and WCW measurements give you the data
needed to determine the growth rate and
doubling time.
Growth Rate and Doubling Time
Calculations
Growth Rate
u = (lnOD2-lnOD1)/T2-T1
Or
u = (lnX2 – lnX1)/T2-T1 (where X=live cell count)
Doubling Time
Td = ln2/u
Live Cell Count: Spread Plate Method
Carefully prepared spread plates for viable cell counts
Prepare serial dilutions and pipet onto the agar plate
Flame sterilize the spreader
Spread the
cells onto
the agar
plate
Optical Density (OD) Measurements
using a Spectrophotometer
Spectrophometers measure Optical Density.
Knowing the OD or, one can determine the
growth rate (u) and from the growth rate, the
doubling time (Td) of the fed-batch culture can
be determined.
Monitoring The Product (Protein)
A uv-visible spectrophometer may be set at 280nm to
determine the concentration of protein in the
media.
Monitoring Growth Conditions
• pH – Often drops as cells grow and divide, if the culture doesn’t get
enough oxygen so that glucose is broken down by glycolysis into lactic
acid which crosses the cell membrane enters the media and creates
an acid environment. If there is plenty of oxygen, glucose is broken
down into pyruvic acid which enters the mitochondria producing H20,
CO2, and energy (ATP and heat).
• Analyate analysis - Glucose concentration measurements using an
analyate analyzer such as a Biolyzer or a Nova, allows us to determine
when glucose has been used up and therefore when to start feeding
methanol for protein production or to determine when lactate is
being produced, a sign of anerobic respiration
• Temperature – Each cell type needs different temperature; Pichia
pastoris require 30 degrees C.
• Dissolved Oxygen – Oxygen is needed to accept protons from the
NADH hydrogen atoms in the mitochondrial electron transport chain.
Process Control
• Head Plate
– Stirrer motor mount (motor controls the agitation rate)
– Condenser (outlet)
– Inoculation port (pump seed reactor contents here)
– pH probe (measures pH of media; negative feedback loop adds
acid or base)
– Thermo well port (put temperature probe in here to measure
temperature; negative feedback loop cools or heats)
– Sparger (bubbles gasses into media)
– Feed bottle (to add glucose, acid or base, methanol)
– DO probe (measures dissolved oxygen in the media; negative
feedback loops control agitation, air and oxygen)
– Harvest port (for harvesting batch)
– Impeller (like a propeller – moves fluid and propelled by motor)
• Computer Controller
Parameters Monitored
(most Process-Control via feedback loops)
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pH (via addition of base or acid)
Temperature (via jackets that heat or cool)
Oxygen (via sparging air or oxygen and agitation)
Rate of Agitation (via need for air or oxygen)
Carbon Dioxide (via sparging)
Feed (via addition of appropriate nutrients)
OD (via spectrophotometer)
Characteristics of Microbial and Mammalian Cell Culture
Parameter
Microbial
Cell Culture
Mammalian
Cell Culture
Growth Rate (u)
Doubling Time = minutes to
hours
Doubling Time = days
Temperature (T)
Great diversity: -0 to +100
degrees (plus/minus 1) C often
by cooling
Most: 37 to 42 degrees C
Control to plus or minus .1
degree C
pH
Great diversity: pH 2-10 (Pichia
= pH 5.8)
Control by adding acid or base
Narrow range: pH 6.8 to 7.2
(CHO = pH 7.2)
Control by sparging CO2
Dissolved Oxygen (DO)
Air or Oxygen sparged
Robust cell walls allow rigorous
sparging
Air sparged
Extremely shear sensitive
use sintered sparger
Agitation Rate
Agitation rates can be >800rpm
Use Rushton impeller
Agitation rates <150rpm
Use maine impeller
Foam
Foam probe, anti-foam agent
required
No foam probe or anti-foam
required
Temperature Control for Mammalian Cell Culture
For mammalian cell culture heating is more critical than
cooling due to slow metabolic rates (doubling time) .
Temperature Probe
Heating Blanket on single wall vessel
Jacketed Vessel for Heating or Cooling
HEATING
– City water
– Heater with recirculation
loop
– Used to heat bioreactors
for mammalian cell culture
at 37 degrees Centigrade
COOLING
– City water
– Chiller with recirculation
loop
– Used to cool fermenters
for microbial cell culture,
e.g. Pichia pastoris or
Escherichia coli
Process-Control Loops
pH Process-Control Loop
DO Process-Control Loop
PID Control
Deviation from Setpoint
20
15
No Control
10
5
PID Control
0
0
5
10
15
-5
-10
Time
20
25